1. Expression of SOD1 G93A mutant in NSC‐34 cells induces cellular stress in a Drp1‐dependent manner
Expression of SOD1 G93A mutant in NSC‐34 cells induces cellular stress in a Drp1‐dependent manner Mitochondria‐specific ROS levels (using indicator MitoSOX), and mitochondrial integrity (using mitochondrial membrane potential TMRM) in hSOD1‐WT‐ and hSOD1‐G93A‐expressing NSC‐34 differentiated cells cultured under serum starvation condition in the presence or absence of P110 (0.25, 0.5, 1, 2 μM/24 h). Results are presented as percent of MOCK (empty vector).Levels of Drp1 were determined in mitochondrial fractions by immunoblotting in hSOD1‐WT‐ and hSOD1‐G93A‐expressing NSC‐34 differentiated cells cultured under serum starvation condition in the presence or absence of P110 (0.25, 0.5, 1 μM/24 h); VDAC, a mitochondrial membrane protein, was used as a loading control. Protein levels were quantified and presented as fold change of hSOD1‐WT.Levels of Drp1 phosphorylation were determined in mitochondrial fractions by immunoblotting using anti‐phosphorylated‐S616‐Drp1 or anti‐phosphorylated‐S637‐Drp1 antibodies in hSOD1‐WT‐ and hSOD1‐G93A‐expressing NSC‐34 differentiated cells under serum starvation in the presence or absence of P110 (1 μM/24 h); β‐actin was used as a loading control. Protein levels were quantified and presented as fold change of hSOD1‐WT.Levels of Parkin and LC3BII autophagy measures were determined in mitochondrial fractions by immunoblotting in hSOD1‐WT‐ and hSOD1‐G93A‐expressing NSC‐34 differentiated cells as in A, cultured in the presence or absence of P110 (1 μM/24 h); VDAC was used as a loading control. Protein levels were quantified and presented as fold change of MOCK (empty vector).Chymotrypsin‐like activity was measured using fluorogenic substrate; Suc‐LLVY‐AMC to measure proteasome activity in homogenates of hSOD1‐WT‐ and hSOD1‐G93A‐expressing NSC‐34 differentiated cells as above in the presence or absence of P110 (1 μM/24 h). Activity levels were quantified and presented as fold change of MOCK (empty vector).Levels of phosphorylated‐eIF2α, XBP1, and ATF‐6 (measures of ER stress) in total fractions were measured by immunoblotting in hSOD1‐WT‐ and hSOD1‐G93A‐expressing NSC‐34 differentiated cells, cultured as above, in the presence or absence of P110 (1 μM/24 h); β‐actin was used as a loading controls. Protein levels were quantified and presented as fold change of hSOD‐1 WT.Data information: Mean, standard deviation, and P‐values are shown. n = 3 performed in (B, C, D, F) duplicate or (A, E) quintuplet; probability by one‐way ANOVA (with Tukey's post hoc test).Source data are available online for this figure.
Amit U Joshi et al. EMBO Mol Med. 2018;10:e8166
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